Heart valve disease, including structural malformations, is the leading cause of mortality in adults. Despite the clinical relevance, the molecular mechanisms required for heart valve formation and function are not well defined. Normal mature heart valve leaflets and supporting structures are differentially composed of diversified extracellular matrix and cell lineages organized to facilitate normal valve function. In contrast, diseased or malfunctioning valves display drastic alterations in connective tissue distribution. We have shown that mature valve supporting structures express the bHLH transcription factor scleraxis and genes associated with tendon cell types. In contrast, valve leaflets express markers related to cartilaginous cell lineages. These specialized cell types are derived from a common population of undifferentiated precursor cells and we have reported that signaling pathways important for valve precursor cell differentiation in vitro are common with tendon and cartilage development. Using an avian in vitro system developed in our lab we have generated data to suggest that ERK1/2-Smad1/5/8 signaling, and activation of downstream scleraxis, are important for valve precursor cell determination and differentiation of a tendon-like lineage that forms valve supporting structures. However further work is required to define these regulatory hierarchies in the development and maintenance of diversified valve structures. Therefore, experiments in avian valve cells in vitro will be used to determine the consequences of altered ERK1/2 and scleraxis function on lineage differentiation in valve precursors and mature valve cells. In addition, generated mice with conditional loss of scleraxis function in vivo will determine the requirements of scleraxis for valve formation and function. The specific aims of the proposed project are to: 1. Determine if antagonistic ERK1/2 and Smad1/5/8 signaling regulates valve precursor cell fate to promote differentiation of tendon- or cartilage-like cell types. 2. Determine if scleraxis is necessary and sufficient to promote tendon-like cell differentiation in valve precursor cells and maintain extracellular matrix organization in mature valve structures. 3. Determine if scleraxis is required for tendon-like cell differentiation and formation of supporting valve structures in vivo. Testing this hypothesis will determine molecular mechanisms important for normal valve development and maintenance. In addition proposed studies will identify regulatory interactions that may underlie congenital valve disease and adult valve degeneration associated with alterations in connective tissue composition and distribution. PUBLIC HEALTH RELEVANCE. Heart valve defects occur in at least 5 million individuals in the United States and the incidence of valve disease is increasing with over 95,0000 surgical procedures to repair or replace malformed valves performed in the US in 2003. However current valve alternatives are limited and often temporary due to inappropriate replacements that fail to grow, remodel, repair or withstand life-long demands. Increased understanding of the processes required for valve development and maintenance has potential clinical significance in identifying genes associated with congenital heart disease and adult valve degeneration, as well as advancing current therapeutic applications.